1. To Optimize a Cell Based Assay for Addiction to Opiates. A tissue culture assay for opiate addiction was devised. Optimal conditions for some assay parameters were determined to enable the assay to be used to screen many compounds for their effects on opiate addiction. 2. To Identify Compounds that Increase CREB Activity as Possible Enhancers of Long-Term Memory. We screened 73,000 compounds by assaying CREB mediated &#946;-lactamase reporter gene expression. We identified 1,800 compounds that potentiated CREB mediated gene expression. Mechanisms of action were determined for some compounds (3-4). A novel class of phosphodiesterase 4 inhibitor was found that is highly potent and highly specific;chemists synthesized 77 derivatives (5) and independently patented these compounds and sold the patent. Four extramural collaborations were established to test some of the most active compounds on long-term memory, and on reactions in tissue culture cells. 3. vnd Homeobox Gene. Expression of the ventral nervous system defective (vnd) homeobox gene initiates neural development in part of the neuroectoderm of the Drosophila embryo that gives rise to some cephalic neuroblasts and 11 of the 31 ventral nerve cord neuroblasts per hemisegment. Vnd also determines the cell types of the 11 ventral nerve cord neuroblasts per hemisegment;each is a different cell type. a. A transgenic Drosophila fly line was constructed that expresses green florescent protein (GFP) only in embryo cells that express vnd. Dissociated cells expressing GFP were purified by FACS. An antibody directed against Vnd was used to purify chromatin fragments by immunoprecipitation. Portions of the purified DNA then were sequenced. More than 1,700 Vnd binding sites in DNA and approximately 1300 genes that are potential targets for Vnd regulation were identified. b. The level of vnd mRNA was decreased in Drosophila embryos by RNA interference and the resulting changes in gene expression were identified. c. A Drosophila neural cell line was transfected with the vnd gene and changes in gene expression due to expression of the vnd gene were determined. d. Nucleotide sequences were identified in the upstream regulatory region of the vnd gene that have been conserved during Drosophila evolution and that regulate vnd gene expression in neuroblasts. Additional constructs of the upstream regulatory region of the vnd gene were prepared with small deletions of nucleotide sequences and the constructs were linked to reporter genes. Transgenic fly lines were obtained for each DNA construct and the expression patterns of the reporter genes were analyzed by immunohistochemistry. Each of the 8 types of neuroblasts studied requires a different combinatorial set of transcription factors for the expression of the vnd gene. Furthermore, transcription factors for 7 of the 8 neuroblasts exhibit cooperativity;i.e., omission of only one of the multiple DNA binding sites for transcription factors required for vnd expression results in no vnd gene expression in those neuroblasts. 4. longitudinals lacking (lola) Gene. The Drosophila lola gene encodes a family of at least 20 proteins that are formed by alternative splicing of RNA and are involved in axon and dendrite pathfinding. All of the Lola proteins have an identical N-terminal region that contains a BTB protein dimerization domain, but each has a different C-terminal region. Seventeen species of Lola protein have different zinc fingers in the C-terminal regions. We tested the hypothesis that isoforms of Lola protein that contain different zinc fingers regulate different sets of genes. Double-stranded RNAs targeting the constant N-terminal region or sequences within the variable C-terminal regions encoded by lola mRNA, were synthesized and each RNA was injected into early Drosophila embryos to decrease the level of the corresponding species of lola mRNA byRNA interference. The effects of RNA interference on gene expression were determined by hybridization of RNA to nucleic acid microarrays. The results show that Lola proteins are transcription factors that regulate gene expression and that each of the 16 lola isoforms tested regulates a different set of genes. 5. Gene Expression by Cells from a Drosophila Neural Cell Line. a. Gene expression was studied by microarray nucleic acid hybridization in undifferentiated ML-Dm-Bg2c2 Drosophila neural cells and after differentiation induced by incubating the cells with a histone deacetylase inhibitor. Four hundred and forty two RNA transcripts were found in ML-Dm-Bg2c2 cells whose levels change after incubation with a histone deacetylase inhibitor. b. Our objective was to use mRNA microarray hybridization to study changes in gene expression evoked by the ecdysone analog, muristerone A, in a Drosophila neural cell line, compared to a non-neural cell line, to identify ecdysone targets that are specific to the neural cells, as well as targets that are independent of the canonical receptor for ecdysone. Exposure of ML-Dm-Bg2c2 neural cells to muristerone A resulted in changes in the levels of 1,936 RNA transcripts. Some (713)of these RNA transcripts change in abundance after exposure to muristerone A in the neural cells but not in the non-neural Schneider S2 cells. Gene expression was studied after 1-10 min exposure of ML-Dm-Bg2c2 to muristerone A, and after reducing the level of the ecdysone receptor, EcR, by RNA interference, and to investigate ecdysone effects that are not mediated by EcR. Translation independent ecdysone targets were identified using ML-Dm-Bg2c2 and S2 cells treated with cyclohexamide 24 hours before exposure to muristerone A. Seven miRNAs were found whose level of expression changed in ML-Dm-Bg2c2 cells after 5 to 48 hrs of exposure to muristerone A but not in cells pretreated with EcR siRNA. Injection of anti-miRNAs complimentary to two of the seven miRNAs affected embryo development. 6. Neuroligans. We have continued our studies of 4 fly genes with homology to vertebrate neuroligins: fnrlg1 (neuroligin 1), fnrlg2 (neuroligin 2), fnrlg3 (neuroligin 3) and fnrlg4 (neuroligin 4). The genes share common structural features of the vertebrate proteins. Loss-of-function mutations in all four genes were generated. Null alleles of fnrlg1 and 2 are homozygous viable and fertile. Adult associative learning and short-term memory are normal in both the fnrlg1 and fnrlg2 mutants. In contrast mutations in fnrlg3 or fnrlg4 are homozygous lethal.